Towards engineered topogenesis of cytochrome b(5) and P450 for in vivo transformation of xenobiotics.

Nature is endowed with catalysts capable of an unprecedented diversity of biotransformations, beyond the capabilities of synthetic chemistries. In a biotechnological context, there is a growing and emerging need to tap this catalytic potential. CYP (cytochrome P450) represents a superfamily of enzymes capable of a diverse array of catalytic activities. Distinct members are engaged in biosynthetic reactions within many organisms, while others have a role in the detoxification of foreign compounds. The latter substrates include medicines, pollutants, pesticides, carcinogens, perfumes and herbicides, representing considerable applied importance for pharmacology and toxicology. CYPs show a high degree of stereo- and regio-specificity for their reactions, which have wide industrial applications. Recombinant CYPs are commonly expressed as active recombinant cytosolic forms in Escherichia coli. However, selective permeability of E. coli to many substrates and products can cause problems with product recovery when using whole-cell systems. To overcome these problems, we have been developing approaches to facilitate export of functional recombinant haemoproteins to the inner membrane, periplasm and the outer membrane of E. coli. Here, we describe the progress in relation to cytochrome b(5) and CYPs.

Kinetic constants of signal peptidase I using cytochrome b5 as a precursor substrate.

A procedure is described for measuring Escherichia coli signal peptidase I activity which exploits an intact precursor protein composed of the alkaline phosphatase signal peptide fused to the full length mammalian cytochrome b5. This cytochrome b5 precursor protein has been extensively characterised and shown to be processed accurately by purified signal peptidase I [Protein Expr. Purif. 7 (1996) 237]. The amphipathic, chimaeric cytochrome b5 precursor was isolated in mg quantities in a highly homogeneous state under non-denaturing conditions. The processing of the cytochrome b5 precursor by signal peptidase displayed Michaelis-Menten kinetics with K(m)=50 microM and k(cat)=11 s(-1). The K(m) was 20-fold lower than that obtained with signal peptide substrates and 3-fold higher than that reported for pro-OmpA-nuclease A precursor fusion. The corresponding turnover number, k(cat), was four orders of magnitude greater than the peptide substrates but was 2-fold lower than pro-OmpA-nuclease A precursor fusion. These results confirm that both the affinities and the catalytic power of the signal peptidase are significantly higher for macromolecular precursor substrates than for the shorter signal peptide substrates.

Human sterol 14alpha-demethylase activity is enhanced by the membrane-bound state of cytochrome b(5).

Human sterol 14alpha-demethylase (P45051; CYP51) catalyzes the oxidative removal of the C32 methyl group of dihydrolanosterol, an essential step in the cholesterol biosynthetic pathway. The reaction is dependent upon NADPH cytochrome P450 reductase (CPR) that donates the electrons for the catalytic cycle. Here we used a recombinant yeast CPR to investigate the abilities of four different forms of cytochrome b(5) to support sterol demethylation activity of CYP51. The cytochrome b(5) derivatives were genetically engineered forms of the native rat cytochrome b(5) core-tail: the soluble globular b(5) core (core), the core linked at its N-terminus with the secretory signal sequence of alkaline phosphatase (signal-core), and the signal sequence linked to the native b(5) (signal-core-tail). The rat core-tail enzyme greatly stimulated sterol demethylation, whereas the signal-core-tail was only marginally active. In contrast, the core and signal-core constructs were completely inactive in stimulating the demethylation reaction. Additionally, cytochrome b(5) enhanced sterol demethylation by more than threefold by accepting electrons from soluble yeast CPR and in its ability to reduce P450. We show that the nature of transient linkage between the hemoproteins and the redox partners is most likely brought about electrostatically, although productive interaction between cytochrome b(5) and CYP51 is governed by the membrane-insertable hydrophobic region in the cytochrome b(5) which in turn determines the correct spatial orientation of the core. This is the first report showing the stimulation of CYP51 by cytochrome b(5).

Histometrics: improvement of the dynamic range of fluorescently stained proteins resolved in electrophoretic gels using hyperspectral imaging.

Most image-based analyses, using absorbance or fluorescence of the spatial distribution of identifiable structures in complex biological systems, use only a very small number of dimensions of possible spectral data for the generation and interpretation of the image. We here extend the concepts of hyperspectral imaging, being developed in remote sensing, into analytical biotechnology. The massive volume of information contained in hyperspectral spectroscopic images requires multivariate analysis in order to extract the chemical and spatial information contained within the data. We here describe the use of multivariate statistical methods to map and quantify common protein staining fluorophores (SYPRO Red, Orange and Tangerine) in electrophoretic gels. Specifically, we find (a) that the 'background' underpinning limits of detection is due more to proteins that have not migrated properly than to impurities or to ineffective destaining, (b) the detailed mechanisms of staining of SYPRO red and orange are apparently not identical, and in particular (c) that these methods can provide two orders of magnitude improvement in the detection limit per pixel, to levels well below the limit observable optically.

A mammalian cytochrome fused to a chloroplast transit peptide is a functional haemoprotein and is imported into isolated chloroplasts.

The small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a major chloroplast stromal protein that is cytosolically synthesized as a precursor with an N-terminal extension, known as the transit sequence or transit peptide (Tp). The Tp is essential for the post-translational uptake of the precursor by the chloroplast. The Tp is thought to influence the conformation of the precursor protein and to facilitate polypeptide translocation across the chloroplast envelope barrier via a Tp-selective translocon. To address these issues we have devised a novel strategy to generate substrate amounts of a chloroplast targeting sequence as a fusion with the chromogenic globular domain of cytochrome b(5) (Cyt). The chimaeric protein is an ideal probe for investigating the conformation of a preprotein and events surrounding protein import into isolated chloroplasts. The Cyt of liver endoplasmic reticulum was fused at its N-terminus with the Tp of the small subunit of Rubisco of Pisum sativum (pea). To enhance its production by clearance from the cytoplasm of Escherichia coli, the chimaera was engineered by further N-terminal linkage of a prokaryotic secretory signal. Expression of this tripartite fusion resulted in mg quantities of the signal sequence-processed Tp-Cyt protein, which was eventually targeted to the membranes. The chromogenic nature of the chimaera and its localization to the bacterial membrane facilitated the biochemical isolation of the precursor in a soluble and functional form. The purified preprotein displayed spectral and enzymic properties that were indistinguishable from the native parental Cyt, implying an absence of observable influence of the Tp on the conformation of the haemoprotein. The chimaeric precursor was imported into the stroma of the isolated chloroplasts in a dose-dependent manner. Import was also strongly dependent upon exogenously supplied ATP. The stromally imported chimaeric precursor protein was processed to a size characteristic of Cyt.

A chloroplast envelope-transfer sequence functions as an export signal in Escherichia coli.

The small subunit precursor of pea ribulose-1,5-bisphosphate carboxylase/oxygenase engineered with prokaryotic elements was expressed in Escherichia coli. This resulted in a dependable level of synthesis of the precursor protein in E. coli. The bacterially synthesised plant precursor protein was translocated from the cytoplasm and targeted to the outer membrane of the envelope zone. During the translocation step, a significant proportion of the precursor was processed to a soluble, mature SSU and found localised in the periplasm. The determined amino acid sequence of the isolated precursor showed that it had a deletion of an arginine residue at position -15 in the transit peptide. Expression of this transit peptide-appended mammalian cytochrome b(5) in E. coli displayed a targeting profile of the chromogenic chimera that was similar to that observed with the plant precursor protein.

Rapid analysis of high-dimensional bioprocesses using multivariate spectroscopies and advanced chemometrics.

There are an increasing number of instrumental methods for obtaining data from biochemical processes, many of which now provide information on many (indeed many hundreds) of variables simultaneously. The wealth of data that these methods provide, however, is useless without the means to extract the required information. As instruments advance, and the quantity of data produced increases, the fields of bioinformatics and chemometrics have consequently grown greatly in importance. The chemometric methods nowadays available are both powerful and dangerous, and there are many issues to be considered when using statistical analyses on data for which there are numerous measurements (which often exceed the number of samples). It is not difficult to carry out statistical analysis on multivariate data in such a way that the results appear much more impressive than they really are. The authors present some of the methods that we have developed and exploited in Aberystwyth for gathering highly multivariate data from bioprocesses, and some techniques of sound multivariate statistical analyses (and of related methods based on neural and evolutionary computing) which can ensure that the results will stand up to the most rigorous scrutiny.

Interaction of human CYP17 (P-450(17alpha), 17alpha-hydroxylase-17,20-lyase) with cytochrome b5: importance of the orientation of the hydrophobic domain of cytochrome b5.

Human CYP17 (P-450(17alpha), 17alpha-hydroxylase-17,20-lyase)-catalysed side-chain cleavage of 17alpha-hydroxyprogestogens into androgens is greatly dependent on the presence of cytochrome b5. The native form of cytochrome b5 is composed of a globular core, residues 1-98, followed by a membrane insertable C-terminal tail, residues 99-133. In the present study the abilities of five different forms of cytochrome b5 to support the side-chain cleavage activity of CYP17 were compared. The five derivatives were: the native pig cytochrome b5 (native pig), its genetically engineered rat counterpart (core-tail), the soluble core form of the latter (core), the core with the secretory signal sequence of alkaline phosphatase appended to its N-terminal (signal-core) and the latter containing the C-terminal tail of the native rat protein (signal-core-tail). When examined by Edman degradation and MS, the engineered proteins were shown to have the expected N-terminal amino acid sequences and molecular masses. The native pig was found to be acetylated at the N-terminal. The native pig and core-tail enzymes were equally efficient at enhancing the side-chain cleavage activity of human CYP17 and the signal-core-tail was 55% as efficient. The core and signal-core constructs were completely inactive in the aforementioned reaction. All the five derivatives were reduced to varying degrees by NADPH:cytochrome P-450 (NADPH-P450) reductase and the relative efficiencies of this reduction were reminiscent of the behaviour of these derivatives in supporting the side-chain cleavage reaction. In the side-chain cleavage assay, however, NADPH-P450 reductase was used in large excess so that the reduction of cytochrome b5 derivatives was not rate-limiting. The results highlight that productive interaction between cytochrome b5 and CYP17 is governed not only by the presence of a membrane insertable hydrophobic region on the cytochrome b5 but also by its defined spatial orientation at the C-terminal.

Glycine-induced extracellular secretion of a recombinant cytochrome expressed in Escherichia coli.

The effect of each of 20 different amino acid supplements to the growth medium of Escherichia coli on the extracellular release of a periplasmic recombinant cytochrome b5 was investigated. Only glycine, and to a lesser extent histidine, stimulated the synthesis of secretory cytochrome b5, as well as its discharge into the medium. Extracellular amounts of cytochrome b5 accrued with increasing concentrations of exogenous glycine and duration of the culture period, in spite of the fact that increasing glycine in the medium progressively inhibited cell growth. For example, 1% medium glycine caused a 50% reduction in bacterial growth, but doubled the periplasmic pool of cytochrome b5 to over 25 micrograms of cytochrome b5/ml of culture at 24 h, a period during which almost all of cellular haemoprotein pool was turned over into the medium. A comparative study of the exportable form of cytochrome b5 with a (non-secretory) cytoplasmic-resident counterpart indicated that the periplasmic cytochrome b5 content was selectively discharged into the medium when less than 1% glycine was present, but, at higher doses, a significant proportion of the additional extracellular haemoprotein was derived from cell lysis. Optimal level of periplasmic discharge of the cytochrome required both active protein synthesis and the presence of a glycine supplement in the medium from the onset of bacterial growth. Phase-contrast and scanning electron microsocopy of glycine-grown Escherichia coli showed that the cells had a 3-7-fold enlarged "eyeball' spheroidal morphology, with a condensed pericircular cytoplasm. The bulk of the volume in such hypertrophied cells consisted of the periplasm; this was reflected by the progressively lowered buoyancy of E. coli cultured with increasing amounts of glycine. The fragility of such cells was apparent by their marked sensitivity to lysis at glycine concentrations above 1%. We conclude that supplementation of E. coli cultures with moderate amounts of glycine substantially stimulates the synthesis of exportable proteins and further enhances their yield by discharge into the growth medium.

Expression, isolation, and characterization of a signal sequence-appended chimeric precursor protein.

This report describes the properties and the functional utility of an unprocessed precursor protein overproduced in Escherichia coli. The precursor protein is from a fusion between DNA sequences coding for the alkaline phosphatase signal sequence and the full-length of rat liver cytochrome b(5). The intact precursor protein accumulated in the membranes represented to over 5% of the total bacterial protein. A procedure involving disruption of the bacterial cells by sonication, isolation of the membranes by differential centrifugation, solubilization with a polar solvent, and ion-exchange chromatography provided milligram quantities of the undegraded precursor in a homogeneous and soluble form. The chimeric precursor protein displayed a characteristic b-type hemoprotein spectrum, identical to that of the native cytochrome b(5). The properties of the precursor protein have been examined by a range of biophysical and biochemical methods. Molecular modeling suggests an amphipathic structure in which a fully preserved soluble core of cytochrome b(5) is terminally bonded by hydrophobic interactions between the amino-terminal signal sequence and the carboxy-terminal membrane anchoring hemoprotein sequence. The precursor substrate was recognized and efficiently cleaved by signal peptidase.

Sheep pancreatic microsomes as an alternative to the dog source for studying protein translocation.

A procedure is described for the preparation of rough membrane vesicles of endoplasmic-reticular origin from the pancreas of sheep. These isolated membranes translocate, process and glycosylate in vitro-translated heterologous proteins in a manner comparable with that exhibited by dog pancreatic microsomes.

Processing of chimeric mammalian cytochrome b5 precursors in Escherichia coli: reaction specificity of signal peptidase and identification of an aminopeptidase in post-translocational processing.

A chimeric precursor interlinked by an arginine residue between the full-length signal sequence of alkaline phosphatase and the eukaryotic cytoplasmic cytochrome b5 was constructed. Expression of the chimeric precursor protein in Escherichia coli resulted in efficient export of spectrally authentic cytochrome b5 into the periplasm [Karim, Harding, Evans, Kaderbhai and Kaderbhai (1993) Bio/Technology 11, 612-618]. On sequencing, the apparent absence of arginine at the N-terminus of the secreted cytochrome b5 implied that the chimera was either miscleaved by signal peptidase or further processed following signal excision by an uncharacterized peptidase. The influence of the N-terminal region of cytochrome b5 on the unusual processing of the chimeric precursor was investigated by engineering a number of variant forms in which the region between Arg+1 and the mature portion of cytochrome b5 was extended and varied. Observations of the in vivo processed patterns of these variant cytochrome b5 forms exported into the periplasm revealed that the absence of arginine was due to neither miscleavage of the translocated precursor by the signal peptidase nor the nature of the early region of cytochrome b5. In fact, the selective excision of the arginine residue occurred subsequent to signal sequence deletion by an aminopeptidase which was sensitive to the metal chelator o-phenanthroline. We show that this aminopeptidase also participates in the trimming of the N-terminal arginine residue of the bacterial alkaline phosphatase to generate the three isoenzymes in the periplasm.

Efficient bacterial export of a eukaryotic cytoplasmic cytochrome.

The soluble core domain of cytochrome b5 of liver endoplasmic reticulum was appended at its amino terminus to full-length alkaline phosphatase secretory signal sequence including the ribosomal binding site. The chimeric precursor gene was placed under the transcriptional control of the native pho promoter in a prokaryotic expression vector. Induction of Escherichia coli by growth in a phosphate-limited medium resulted in abundant synthesis of cytochrome b5 as detected spectrophotometrically and by visual transformation of the bacteria to a pink color. The signal-appended cytochrome b5, but not the corresponding signal-deficient derivative, was translocated across the bacterial inner membrane and processed to yield authentic, haem-assembled cytochrome b5 within the periplasm. The eventual processing of the chimeric cytochrome b5 precursor was unusual regarding the known reaction specificity of signal peptidase. The exported, mature haemoprotein was biochemically indistinguishable from its native mammalian counterpart. At peak induction, approximately 6 mg of correctly matured cytochrome b5 per liter of culture was exported. This amount of cytochrome b5 constituted 6% (w/w) of the periplasmic protein. The appearance of the exported apo-cytochrome b5 preceded the formation of holo-protein. Thus the eukaryotic cytoplasmic protein was efficiently exported from E. coli and post-translocationally modified to generate a functional haemoprotein in the periplasm.

Gene-dose-dependent expression of soluble mammalian cytochrome b5 in Escherichia coli.

A synthetic structural gene encoding a mammalian cytochrome b5, carrying an optimised ribosomal binding sequence, was tandemly polymerised ranging from one (n = 1) to six (n = 6) gene copies. The gene, placed in p lambda-ncyt under the control of the lambda PL promoter, transcribed mono- to hexahomocistronic mRNA, expressing one to six copies of cytochrome b5. The expressed levels of cytochrome b5 in Escherichia coli p lambda-ncyt corresponded linearly with the gene dose when up to five copies were present; saturating build-up of the recombinant protein was reached at six gene copies. Cells bearing p lambda-6cyt produced 75 micrograms cytochrome b5/ml of unit optical density at 600 nm culture, constituting 55% of the soluble bacterial protein. The recombinant protein accumulated predominantly in a haem-deficient, apoform, together with lesser amounts of the holocytochrome b5. Whereas the overall expressed protein (apo and holo forms) was gene dose dependent, there was an inverse relationship between holocytochrome b5 production and gene dose. Incubation of the thermally induced bacterial lysates with exogenous haem a converted all of the soluble apocytochrome b5 into holocytochrome b5 that was spectrally indistinguishable with its native counterpart. Culture supplementation with the likely metabolic precursors of haem synthesis, 5-aminolevulinic acid, glycine/succinate or glutamate, significantly alleviated the protoporphyrin deficiency during hyperproduction of cytochrome b5 in E. coli.

A pink bacterium as a reporter system signaling expression of a recombinant protein.

We describe a set of expression vectors, pEX-PINK/0-3, for high-level production of (un)fused target proteins. The vectors incorporate a 'pink' reporter element, which signals in vivo the expression status of a target gene. A target sequence is cloned between the lambda PL promoter and the downstream mammalian cytochrome b5 gene. Thermo-induction drives transcription of a dicistronic mRNA from which the target protein and cytochrome b5 are independently and concurrently synthesized. Positive expression is indicated by visual transformation of bacteria from a grey/translucent to a bright pink color derived from tandemly expressed holocytochrome b5. The signal can be monitored in vivo spectrophotometrically.

Co-expression of a precursor and the mature protein of wheat ribulose-1,5-bisphosphate carboxylase small subunit from a single gene in Escherichia coli.

The cDNA encoding a precursor of wheat ribulose-1,5-bisphosphate carboxylase/oxygenase was inserted in-phase with prokaryotic expression elements in four different vectors. Five expression vectors encoding the small subunit precursors were cloned in Escherichia coli. None of these constructs expressed detectable amounts of the precursor protein, but all directed synthesis of the mature small subunit. The expression of the small subunit was a consequence of an independent, intragenic Shine-Dalgarno sequence optimally located upstream from an ATG specifying the first codon of the mature small subunit portion in the precursor transcript. Similar internal translation signals have been identified in the nuclear-encoded cDNAs of the small-subunit precursors of numerous higher plant genes. The 5' end of the wheat small-subunit precursor was linked with a consensus E. coli DNA sequence such that the modified gene encoded a partial hybrid precursor carrying four additional residues at its amino terminus. The resultant construct, pEI-W3, directed abundant synthesis of both the partially hybrid small-subunit precursor and the mature small subunit, constituting as much as 10% of the total bacterial protein. The bacterially synthesized small subunit precursor was purified to homogeneity. The authenticity of the recombinant protein was verified by its size, immunological properties, amino-terminal sequence, and amino acid composition.